Limitations of Common Molecular Markers in Fungal Biodiversity Analysis and the Benefits of Their Synergistic Use

High-throughput sequencing of the Internal Transcribed Spacer (ITS) regions is the primary method for estimating fungal diversity from environmental DNA. However, reliance solely on ITS markers is complicated by its high variability in sequence length and the presence of multiple variants within a single genome, which can bias diversity estimates. This study compares the performance of the ITS1 and ITS2 molecular markers with five alternative markers targeting protein-coding genes using a defined mock community of 413 fungal species from 32 orders. We show that none of the markers provided a reliable estimate of fungal diversity and two failed to produce sufficient data. While the ITS2 marker showed the highest proportion of individual species detections at 75.3%, a multi-marker approach significantly improved this result. Combining the top three markers (ITS1, ITS2 and elongation factor 1-alpha gene) increased this number to 92.2%. This approach also provided a crucial corrective for relative sequence abundance (RSA) estimates, as combining markers helps to offset the individual biases of each of them resulting in a more balanced picture of the community composition. Although the ITS region remains a cornerstone in fungal metabarcoding studies, we recommend combining both ITS1 and ITS2 markers with at least one alternative DNA marker to achieve more accurate species richness and RSA estimates. The broader application of these alternative markers will depend on the continued development of comprehensive, curated reference databases.